Aquafeed Vol 13 Issue 2 2021

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Vol 13 Issue 2 April 2021

AQUAFEED Advances in processing & formulation An Aquafeed.com publication

IMPROVING EXTRUSION PROCESSING Phytogenic and plant-extract applications Functional lipids in aquafeeds DNA-based analyses for aquafeed transparency Published by: Aquafeed.com LLC. Kailua, Hawaii 96734, USA www.aquafeed.com info@aquafeed.com


FE E D AND B IOFU E L

ADVANCE D FE E D PRODUCTION TECHNOLOGY THE NEW T WIN SCREW EX TRUDER

TWIN SCREW EXTRUDER ANDRITZ Twin Screw Extruders developed for increased demands in aqua feed. For aquatic feeds, the Twin Screw Extruder ensures complete utilization

of starch; allowing for higher flexibility in the formulation and enabling a higher feed conversion ratio. Many variations in formulas can be controlled and handled without having to change the screw configuration.

ANDRITZ FEED & BIOFUEL A/S ⁄ Europe, Asia, and South America: andritz-fb@andritz.com USA and Canada: andritz-fb.us@andritz.com ⁄ andritz.com/ft

Find out how our vast expertise and patented aqua feed and pet food processing technologies can feed the future of your business at andritz.com/ft.


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AQUAFEED

VOL 13 ISSUE 2 2021

Contents

SUSTAINABLE MANUFACTURING OF SHRIMP FEED 17 How shrimp feed processing with twin screw extrusion offers benefits as a sustainable manufacturing method.

FEATHER MEAL IN SHRIMP FEEDS 30 Nutritionally improved feather meal can reduce the rate of fishmeal in shrimp feeds without affecting productivity.

ENHANCING GROWTH PERFORMANCE OF TILAPIA 39

DNA-BASED ANALYSES FOR TRANSPARENCY 48

Phytogenic feed additives can improve fish tolerance to environmental and inflammatory stress and thus enhance growth performance.

How NGS analyses can screen raw materials and ensure the origin of the ingredients and avoid unwanted species in aquafeeds.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 2 2021


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AQUAFEED

VOL 13 ISSUE 2 2021

Contents 6

Interview

9

News Review

14 Vacuum or pressure:

6

Which is the better density control solution?

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Sustainable manufacturing of shrimp feed with twin screw extrusion

21 High moisture extrusion - a promising new method for making aquafeed with unique properties

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F ish protein concentrate can partly replace water as plasticizer in the fish feed extrusion process

28 Pigmentation and growth performance on seabream and shrimp

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ecent trials show large potential for nutritionally R improved feather meal in shrimp feeds

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All-natural: Immune protection, enhanced performance

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How to enhance growth performance of tilapia Plants extracts in aquafeeds: Standardization as a key parameter Phytogenic feed additives to counteract mycotoxin impact on fish health

Improved DNA-analyses as tools to cover the need for evidence-based transparency in the aquaculture industry

Columns 36 Albert Tacon – Disorders in lipid and fatty acid nutrition

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48

Calendar of events

To read previous issues in digital format or to order print copies, visit: http://www.aquafeed.com/publications/aquafeed-magazine/

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 2 2021


AQUACULTURE

Share Our Vision

A AQ036-20

Species-specific solutions for a sustainable and profitable aquaculture At Adisseo, we offer species-specific nutrition and health solutions to aquaculture customers around the world. There is a lot to gain by optimizing your feed additive strategy. Our aqua experts are passionate to help you find out how to increase your productivity and profitability. We look forward to sharing our vision with you!

www.adisseo.com


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Yeo Keng Joon is Chairman and Managing Director of Bharat Luxindo Agrifeeds Pvt. Ltd. The company was the first to introduce complete formulation fish feed in India and is now one of the major players in India that commercializes fish and shrimp feed.

INTERVIEW AQUAFEED: What has been your journey in aquafeeds? How did you get to where you are today? YKJ: In 1988, I was appointed to start the aquaculture business in the Gold Coin group of companies as new business development for a company focused thus far on pig and poultry feeds. Starting with a shrimp feed plant in Johor Bahru, Malaysia, we gradually grew the business with plants in Jurong, Singapore; Bekasi, Indonesia; Medan, Indonesia; Manila, Philippines; and Hatyai, Thailand. It was a joy to be able to build up a team of professionals in all these operations and very satisfying to see the business volume grow by significant leaps and bounds. Breaking new ground into a new industry was an exciting challenge for our young team

with Yeo Keng Joon and many have progressed very well since then. I left the Gold Coin group in 1998 for new challenges, having had the satisfaction that the aquaculture business had matured over the ten years that I was leading it. What is the relationship between Global Group, Singapore and Bharat Luxindo Agrifeeds? YKJ: In 1997, some of the managers in Gold Coin Indonesia left to start an aquaculture business in Medan. I was invited to join them in Global Aquaculture Indonesia which started producing shrimp feed and extruded floating feed for tilapia farming in North Sumatra. The group started its second venture with a shrimp feed plant in Jakarta. In 2006, we were invited

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to be a partner in Laila Global Pvt Ltd, to produce shrimp feed in Andhra Pradesh. The Indian partner had been using shrimp feed imported from Global Aquaculture Indonesia and wanted a manufacturing base in India. Unfortunately, the business floundered and we then set up Bharat Luxindo Agrifeeds Pvt Ltd to take over the business. We decided to focus on pelleted fish feed for the Indian carp (Rohu) which is largely using very traditional feeding of deoiled rice bran powder and groundnut cake. AQUAFEED: The company has come a long way since it first expanded into India in 2006. What is the scope of the Bharat Luxindo Agrifeeds now? YKJ: Traditional fish farming is a very large market in India and the potential to convert these farms to use pelleted fish feed is tremendous. As with changing the traditional mindset, it is a very challenging task and we continue to use various tactics and strategies to convince the farmers slowly but surely. Demonstrating our feed performance and sharing of technical data is key to overcome these challenges. We have also gone into producing Rohu fingerlings with our own broodstock selection so that our customers have access to a good supply of fingerlings. AQUAFEED: In January, Bharat Luxindo Agrifeeds Pvt. Ltd. inaugurated its new feed mill in Chittavaram, Narasapur. How much capacity did this add, and what type of aquafeeds does it produce? YKJ: We have the capacity to produce 2,000 tons of pelleted fish feed in our new plant in Narasapur. We are focused on the Rohu and Katla fish industry and are also into extruded floating feed business through our contract production arrangements. AQUAFEED: How were you able to do this during the pandemic? YKJ: We have a very dedicated team of local Indian managers who have been working with us in the construction of our new plant. Many of these managers have been with the company for more than ten years and understand the business and share our corporate culture of good teamwork. We have regular Zoom meetings to ensure the smooth operations of our business.

Traditional fish farming is a very large market in India and the potential to convert these farms to use pelleted fish feed is tremendous. As with changing the traditional mindset, it is a very challenging task and we continue to use various tactics and strategies to convince the farmers slowly but surely. AQUAFEED: Bharat Luxindo Agrifeeds has put a lot of effort into helping fish farmers in India become more efficient, particularly in Rohu farming. How are you going about this? YKJ: As mentioned previously, converting the traditional mindset of using raw materials in powder form to be replaced by nutritionally balanced pelleted feed is a significant challenge. We have slowly but surely convinced many farmers on the more scientific and progressive use of pellets which will result in faster growth and shorter harvest cycles. More importantly, the raw materials pollute the pond environment and contribute to bacterial growth in the pond bottoms and water channels. This pollution had to be more widely publicized so that the community is aware and more urgent combative steps are taken to overcome this. We continue to adhere to our objective of introducing best practices in aquaculture farming to the fish industry and community in India. AQUAFEED: What do you see as the future of aquaculture in India, and the potential for aquafeed? YKJ: There is tremendous potential for the modernistic development of aquaculture in India. With the large population that India currently has, the per capita consumption of fish will definitely increase as the economy grows and the country develops. Fish protein is one of the most healthy meat proteins that are

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currently available and more publicity of the benefits of fish meat will see the potential for aquaculture increase significantly in the next few years. AQUAFEED: What has been the biggest change you have seen in aquaculture in Indonesia and India over the years? YKJ: The introduction of best practices in aquaculture will continue to impact the industry in the next few years. Better water management will enhance the yield of the industry together with the use of nutritionally balanced fish feed pellets. AQUAFEED: On a more personal note, philanthropy is a prominent part of your life. Notable is your role in setting up the National University of Singapore (NUS) Business School Alumni Association Bursary Fund, and your efforts to encourage entrepreneurship amongst youths in Singapore. Why is it so important to you to support those less fortunate than yourself? YKJ: Each and every one of us should continue to share our blessings and give our help and support

to the less fortunate. I am very focused on helping needy students so as to enable more upward social mobility. To be able to help and make a difference in someone’s life is very enriching and joyful. Giving and seeing the impact of our giving is very empowering. Encouraging entrepreneurship in Singapore is my way of sharing and giving back to the community based on the work experience that I have had in the countries of Southeast Asia and now in India. There are tremendous opportunities for entrepreneurship in aquaculture in India. There is tremendous scope in the integration of marketing activities. For example, marketing and selling of branded packaged fish fillets marinated with local spices and sauces – microwaved dinner ready packages for the huge urban population in the Indian cities. Managing the supply chain from fish ponds to the supermarket chains and retail outlets offer huge entrepreneurial opportunities in India.

CLEAN FEED. CLEAN WATER. Wenger Extrusion Solutions for RAS Feed Production Wenger innovative extrusion solutions deliver clean, durable, nutritional feeds specifically designed for the most efficient RAS operations. Feeds produced on Wenger systems maintain their integrity better and longer, for clean and clear water. So you feed the fish, not the filter. Learn more about the Wenger RAS advantage. Email us at aquafeed@wenger.com today. PHONE: 785.284.2133 | EMAIL: AQUAFEED@WENGER.COM | WENGER.COM USA

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NEWS REVIEW Highlights of recent news from Aquafeed.com Sign up at Aquafeed.com for our free weekly newsletter for up-to-the-minute industry news

Skretting invests $30.1 million in new projects

BioMar acquires majority shares of Viet-Uc After comprehensive due diligence, BioMar and Viet-Uc have signed an agreement for BioMar to acquire the majority of Viet-Uc feed business. Through this acquisition, BioMar partners with one of the leading seafood groups active in shrimp hatcheries, fish hatcheries and shrimp farming in Vietnam. The ambition is to grow the market for high-end feed products focusing on sustainability, traceability, quality and performance.

The company plans to construct a new factory in Vietnam in 2021 as part of its commitment to aquaculture in the country and the broader Southeast Asia region. With an investment of €24 million, the new factory will supply the growing market in the Mekong Delta and beyond with an annual capacity of 100,000 tons. Skretting is also building a new R&D shrimp facility in Ecuador to complement Skretting's network of Aquaculture Research Center (ARC) network. The new R&D center will comprise fully equipped laboratories and state-of-the-art experimental units to carry out tests under controlled conditions. In addition, greenwater tanks will ensure maximum applicability in production conditions.

Nutrition Technologies opens new industrial-scale insect factory in Malaysia The Singapore-based company opened its first industrial-scale insect protein factory that will produce 16,000 tons of insect products per year. The company also closed a recent

funding round of $5m, led by Hera Capital, and supported by existing investors, Openspace Ventures and SEEDS Capital that will be used to fund new R&D projects as well as prepare the company for entry into new markets in Southeast Asia.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 2 2021


NEW ON THE MARKET New counterflow recovery unit allows up to 75% energy recovery Geelen Counterflow developed the Counterflow Recovery Unit, a top-down vertical heat exchanger in stainless steel that recovers up to 75% of the energy and water from a counterflow dryer’s exhaust air while avoiding the excessive fouling by fines and fats that often make the use of industrial heat exchangers so difficult. Together with an electric heat pump and heat exchangers for the drying air, it can eliminate the need for fossil fuels for drying extruded aquafeeds.

Brabender introduces inline viscosity measurement and process control The company introduced the Convimeter II for viscosity measurement and process control in production for the development of liquids and pastes. It opens the door for many measurement methods previously reserved for offline rheology to enter production environments. This supports the development

of manufacturing processes and enables large-volume testing on a production scale.

Bühler, Premier Tech introduce new fully automated packaging equipment The new fully automatic OML-1060 packaging solution is a more affordable iteration of an existing high-end solution. It can easily handle free-flowing granular material such as animal feeds and 20 to 50 kg bags at a speed of up to 600 bags per hour. Its small and compact footprint optimizes floorspace and its lean design eases operation and maintenance. It requires very limited operator interaction, enabling better working conditions.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 2 2021

BIOMIN introduces mycotoxin solution in Asia Pacific region

Biomin introduced its newest mycotoxin risk management solution, Mycofix Plus 5.Z with ZENzyme, in select markets across the Asia Pacific region. It is an innovative, all-in-one feed additive providing nextgeneration mycotoxin risk management for breeding animals and their offspring. ZENzyme is the first and only purified enzyme that degrades zearalenone (ZEN) fast, specifically and irreversibly into non-toxic and non-estrogenic metabolites.


NEW ON THE MARKET New Bühler remote service solution sets new standard for manufacturing efficiency

Romer Labs introduces an on-site quantification solution for mycotoxins

The all-new BühlerVision provides advanced remote service and maintenance, powered by the latest smart glass technology and augmented reality software. Customers are connected to Bühler experts remotely, which significantly speeds up reaction time, reduces travel time and the costs and safety risks associated with on-site service visits.

The company unveiled the AgraStrip® Pro WATEX® test system for the rapid and simple on-site quantification of four commonly regulated mycotoxins (total aflatoxin, deoxynivalenol, total fumonisin and zearalenone) in a variety of agricultural commodities. The new system makes mycotoxin testing more efficient, simplifying workflow and allowing for optimal time management. The entire test procedure is completed in ten minutes, with the assay itself taking only four minutes.

Dr. Eckel introduces new product against endotoxins Following two years of development and specially developed to defend against dangerous endotoxins, Anta®Catch promises maximum effectiveness and comprehensive protection and gets the threat posed by endotoxins under control for farmers and producers. MAX.

393.31 [9990]

MAX.

391.31 [9939]

15.00 [381]

Ă12.00 [305]

MIN.

29.19 [741]

F085 SHIMPO

36.91 [937]

31.19 [792]

MIN.

67.28 [1709]

39.00 [991] 101.44 [2577]

30.38 [772]

ALL FROM A SINGLE SYSTEM

BIN Inlet

P.O. Box 8 100 Airport Road Sabetha, KS 66534, USA Phone: 785-284-2153 Fax: 785-284-3143

30.00 [762]

19.16 [487]

64.83 [1647]

108.59 [2759] DCC Inlet

End of Head

CYL. Disch.

15.00 [381]

With Extru-Tech’s ADT (Advanced Densification Technology), the possibilities are far reaching. ADT technology gives you the option to produce sinking feeds with excellent consistency and density. That same ADT technology can produce floating

FROM SINKING TO FLOATING

feeds with high protein characteristics … all from a single extrusion system. 269.88

In the aquafeed business, you either sink [6855] or swim. Contact Extru-Tech today at 785-284-2153 or visit us online at www.extru-techinc.com

284.00 [7214]

278.03 [7062] 1.93 [49]

extru-techinc@extru-techinc.com www.extru-techinc.com

199.38 [5064]

18.00 [457]

1.00 NPT

P.O. Box 8 • 100 Airport Road • Sabetha, KS 66534, USA Phone: 785-284-2153 • Fax: 785-284-3143

12.56 [319]

51

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15.88 [404]

24.59 [625]

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108.28 [2750]

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80 NORGREN

0

88.00 [2236]

160

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2.00 NPT [WATER] 3/4 NPT

2.00 NPT

53.25 [1353]

66.50 [1689]

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 2 2021 102.13 [2594] 111.12 [2822] 195.72 [4971]

52.19 [1325]


PRODUCT FOCUS Fritsch introduces fast, accurate knife mill for sample preparation Since feed and raw materials are often composed of heterogeneous substances, in recent years, a wide range of sample preparation methods has been developed for feed analysis. Fritsch now offers an industrial-grade knife mill, the Knife Mill Pulverisette 11. Each subsample taken from any location in the grinding vessel is representative of the original sample and therefore ensures accurate, significant analysis for a wide range of different materials, regardless of whether the sample is dry, wet, soft, medium-hard, fibrous or oily. The advantages of the Pulverisette 11 are that it is easy to clean, the parts in contact with the sample are autoclavable, and it has variable speed settings and turbo function. With the P-11Control software, the mill can be controlled via the USB port and up to 20 SOPs can be edited, saved and managed directly on the connected laptop via drag and drop.

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Vacuum or pressure: Which is the better density control solution? Rob Strathman, Famsun-USA Design and Engineering

Sinking aquafeed pellets allow bottom-dwelling aquatic species to quickly locate and consume their meal before it disintegrates into water pollutants or is otherwise consumed by opportunistic scavengers. Cooking and compressing the feed ingredients into sufficiently dense pellets during extrusion, however, is often challenging. There are multiple operational techniques and pieces of hardware employed to maximize the bulk density of sinking products. However, two extruder add-on devices can provide variable density control and are commonly used within the industry. One such control system uses a mid-barrel vacuum system, while the other uses an end of the barrel pressure chamber. But the question we are frequently asked is, which is the most effective? The underlying operating principle of both systems is to alter the melt's water vapor pressure force as it passes through the extruder die. The melt is the raw material after it transitions from a crystalline structure into a viscoelastic fluid near the extruder die. Water vapor pressure is a function of the melt temperature, which ranges from 100 to 150°C for most extruded aquafeeds. As can be found on a saturated steam chart, water within this temperature range

experiences a corresponding vapor pressure of 1.0 to 4.8 bar. The water vapor pressure, not the commonly misconceived die pressure, is the driving force behind product expansion. Therefore, it may seem logical that dense feeds would require a low melt temperature, while highly expanded floating feeds could operate at higher temperatures. Although reducing the melt temperature is a proper technique for controlling expansion, if it is too low, it often results in more significant challenges: uncooked starch and easily broken pellets. This set of counterintuitive conditions are the compelling reasons why these systems are beneficial and often necessary when extruding certain sinking feed products. The Vacuum-Density Control System (V-DCS), see Figure 1, functions by allowing high levels of both thermal and mechanical energy to be applied earlyon in the extruder barrel. A vacuum port, located more than halfway down the barrel, then degasses or removes a portion of the high-energy water vapor from the melt. This approach first gelatinizes the starch and then cools the melt before it exits the die. The result is a denser and more durable pellet. Pressure-Density Control Systems (P-DCS), see Figure 2, use compressed air to pressurize a die encapsulating

Figure 1. An extruder equipped with a Vacuum-Density Control System.

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Figure 2. An extruder equipped with a Pressure-Density Control System.

consistency before reaching the vacuum port, making the product more difficult to become airborne and far less likely than fines to be extracted. The typical melt moisture required when using a vacuum system is in the neighborhood of 29-33%.

Figure 3. Pellets from a P-DCS. Pressure: Left = None; Right = 2 bar. Bulk density: Left = 600 g/L; Right = 700 g/L.

chamber to a selected level between 0.5 and 2.0 bar. The altered environment sufficiently raises the boiling point of water, preventing the moisture within the pellet from vaporizing and hindering the product's expansion. This approach also ensures a high degree of starch gelatinization, pellet durability, and an optimum density. Product photos in Figure 3 illustrate the differences in pellet porosity, texture, color and density when the pressure chamber is operating at 0 and 2 bar.

Operational considerations The vacuum and pressure-based systems are arguably equally effective at manipulating the bulk density of aquafeed pellets. Yet, operationally, they both offer a unique set of conditions that must be considered before making a selection. Processing moisture A significant drawback of the vacuum systems is their tendency to pull raw material from the barrel. Hence, these systems are equipped with an integrated stuffing screw (Fig. 4) and a particulate separation system (not shown). One necessary means of minimizing the number of fine particles removed is to use large volumes of water during production. The added moisture ensures the raw materials are in a "dough-like"

Dryer energy requirements The V-DCS operates at melt moistures that are about 3-6% greater than a P-DCS system. Table 1 compares the extrusion moisture differences between these two technologies and illustrates the impact of a 3% difference in melt moisture on the dryer's workload. In this example, the dryer’s evaporative load and energy requirement when using a V-DCS are approximately 22% greater than when using a P-DCS system. It is important to note that in existing production lines, where a dryer is a capacity bottleneck, selecting a pressure system may also significantly increase throughputs. Potentially presenting a hefty ROI to the operation. Screw configurations – Impact on capacity and starch gelatinization For some manufacturers, the P-DCS's ability to produce both floating and sinking products with just one screw configuration is a significant benefit. The V-DCS, however, requires a specific screw configuration for sinking feeds, which is not optimal for floating feeds. Thus, a screw configuration change is necessary to optimize the capacity of both products. The screw configuration required for the V-DCS is broken into two distinct sections: the cooking zone and the forming zone. The cooking zone is a short region in front of the vacuum port, while the forming zone is located between the vacuum port and the die (Fig. 1). The short cooking zone can leave some

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Table 1. Impact of extrusion moisture on drying.

Add-on Density Control System

Extruder Parameters

Dry Mix Rate Steam Flow Rate Water Flow Rate Melt Moisture Extruder Discharge Rate

Kg/hr Kg/hr Kg/hr % Kg/hr

4,000 320 665 27.0% 4,985

4,000 320 880 30.0% 5,200

Dryer Parameters

Impact of Extrusion Moisture on the Dryer's Evaporative Load

Target Final Moisture Dryer Discharge Rate Evaporative Load @ Drying Efficiency Energy Required

% Kg/hr Kg/hr MJ/Kg MJ/Kg

9.0% 4,000 985 2.92 2,876

9.0% 4,000 1,200 2.92 3,504

Figure 4. An extruder barrel with a stuffer screw and vacuum port.

starch uncooked, potentially causing pellet durability issues. This becomes more problematic when making larger diameter pellets, which are more susceptible to damage caused by impact and attrition forces during handling. The P-DCS does not require the forming zone, thus typically cooks the starch more thoroughly.

Pressure Vacuum Diff.

4.3%

21.8% 21.8%

Die change/Diet change-over downtime The Pressure-DCS (Fig. 5) has an operational challenge of its own, namely the extra downtime required at each diet change-over to remove the chamber and gain access to the die. The chamber contains several additional fasteners that are time-consuming to remove and re-install, adding approximately 10 minutes to each change-over.

Selecting the right density control system Both add-on density control systems serve their intended purpose of controlling pellet density very well, but the pressure version does offer multiple operational advantages. Yet, not all sinking aquafeeds are designed equally and there are applications where the vacuum version may be more desirable. For example, those products requiring high levels of water stability may benefit from the higher moisture conditions required by the V-DCS, thus leveling the playing field and making the vacuum version an option to consider. Ultimately, the product design characteristics must be the first consideration when making a purchasing decision and operational factors should be a distant second.

More information: Rob Strathman President Famsun-USA Design & Engineering, USA E: Rob.Strathman@Famsun-USA.com Figure 5. Famsun’s pressure-density control system.

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Sustainable manufacturing of shrimp feed with twin screw extrusion Hadrien Delemazure, Clextral

In the global movement towards sustainable food sources, shrimp farming presents an important opportunity for aquaculture producers. Although the COVID-19 pandemic limited farming and temporarily reduced demand, worldwide consumption of shrimp is projected to reach $7.65 billion by 2023 (Global Shrimp Market 2019-2023). Consumers have come to value this small crustacean’s high nutritional impact and health benefits and are adding it regularly to their diet. Shrimp is a known source of the trace element selenium

with antioxidant attributes that have been linked to increased immunity. Yet, with this market opportunity comes challenges: • How to produce high-quality feed that supports sustainable shrimp health and growth. • How to optimize Feed Conversion Ratios (FCRs) and minimize waste that negatively impacts the environment. • As the industry faces scrutiny about traditional feed ingredients (fishmeal and fish oil), combined

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with periodic scarcity and resulting high ingredient cost, how to incorporate alternative and plantbased ingredients. • How to manufacture sustainably using fewer natural resources (energy, water, etc.). In this article, we will discuss these challenges and how shrimp feed processing with twin screw extrusion offers benefits both as a sustainable manufacturing method and also by producing shrimp feed that contributes to sustainable growth and environment conservation.

Processing for maximum nutrition and growth Feed quality directly impacts the size and quality of harvested shrimp, however, shrimp feeding practices vary widely depending on the species and resources of the grower. For healthy growth to optimal size, shrimp require a nutritionally balanced diet, rich in protein and fats. For best absorption, feed ingredients must be properly managed, mixed and processed to fully combine the proteins and fats and integrate the carbohydrate components (starches) while protecting delicate enzymes and nutrients (vitamins and minerals). This is where the twin screw extruder excels. As ingredients are transported by the continuous, co-rotating screw action through the extruder barrel, the pressure, temperature and moisture can be precisely and independently controlled in every section. This complete control of production parameters ensures optimized starch gelatinization

and complete protein cooking for best absorption. The intermeshing screw configuration of the extruder is key to positive transfer and uniform mixing when processing feeds with high-fat content. Typically ingredients are processed in a preconditioner before extrusion, as this pre-processing step ensures homogenous distribution of the ingredients and initial starch gelatinization and denaturing of the proteins. The result of these combined processes, preconditioning and extrusion, is high-quality feed with excellent digestibility and textural qualities. Multiple studies by industry experts have shown that feeding high-quality feed to young shrimp significantly benefits their health and growth.

Processing for optimized Feed Conversion Ratio (FCR) with minimum waste Whether shrimp are farmed in ponds or open nets,

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virtually all are fed with commercial feed. Research has shown that feeds designed for the specific nutritional requirements for the species ensure optimal FCR, reduce waste and minimize farmers’ costs. The twin screw extruder has the flexibility to process a variety of feed types on the same machine by simply modifying the ingredients, production parameters and die configuration. Recipes and profiles are typically preset for access at the operator control station for the quick changeover on the production line. Clextral extruders offer a density control system (DCS) that controls the thermal energy to be added or removed to adjust the feed pellet density, a benefit for shrimp feed production. This fully automatic tool increases the melt densities up to 750 g/L to achieve feed with high water stability and fast sinking properties. Water stability of more than 24 hours is key for optimizing feed consumption, as shrimp feeding activity is highest at night, while feed distribution is normally done during daylight hours. The ability to match feed characteristics with the shrimp’s habits is key to eliminating food waste and maintaining a healthy aquatic environment. The feed size must also match the species’ mouth capacity. Clextral’s production dies for shrimp feed start at 0.5 mm. In order to form this small feed size, 100% of the mix granulometry must be less than one-third of the die holes, so in this case, 165 µm. This is achieved with a specialized grinding and pulverizing process. With die changes, the same extruder can process feed with perfectly calibrated dimensions from 0.5 to 3.0 mm and Clextral offers a range of die designs and cutters for this purpose. This flexibility enables specific matching for shrimp feeding patterns to ensure optimal feed consumption at every stage of growth. Feed storage can also be a factor, especially for smaller farmers. Shrimp feed pellets produced by twin screw extrusion have proven to offer high shelf stability with minimum degradation due to their superior density and compact behavior.

Processing with plant-based ingredients It has long been believed that shrimp feed must contain both fishmeal and fish oil for proper nutrition. However, with the increase in world aquaculture, these ingredients are often in short supply, resulting in higher costs. This has led researchers to explore new

ingredients to obtain optimal nutrition. A recent study at Georgia State University demonstrated that shrimp feed based on soybean meal with added artificial chemostimulants was consumed as well as formulas that included krill, a known attractor. Digestibility and palatability are top concerns when developing new shrimp feed formulations. Clextral, as a major player in extruder technology for feed manufacture, has developed innovative techniques and technology for processing original recipes with raw materials that include pulses, proteins, insects, as well as processed animal proteins and seaweeds.

Sustainable manufacturing Much has been discussed about sustainable manufacturing, in fact, the US Environmental Protection Agency (EPA) defined it as “the creation of manufactured products through economically-sound processes that minimize negative environmental impacts while conserving energy and natural resources.” If we look at twin screw extrusion processing, it is evident that this technology is well-aligned with this definition of a sustainable manufacturing operation. Let’s take a closer look. The twin screw extruder enables optimized processing with less waste. As a closed and continuous reactor, the extruder enables precise control and regulation of all parameters. Clextral preconditioners’ patented Advanced Filling Control (AFC) system is designed to control the filling ratio and residence time inside the preconditioner unit. The high output design of the Evolum+ extruder produces up to 40% greater volume than other systems, to generate more product at a reduced capital expenditure. The DCS system recycles fines and condensates from the extruder back into the preconditioner, forming a perfect closed loop system with no waste. Efficient use of water in extrusion results in lower requirements and less effluent produced. In both the preconditioner and extruder, adjustable steam injection points are located for maximized absorption during processing. Energy requirements are also lower due to several key features. In the preconditioner, steam injection can efficiently generate product temperatures of over 96˚C while limiting energy loss. At the extruder, a high torque gearbox generates maximum screw speed and

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mechanical energy. Independent barrel modules are precisely heated or cooled by Clextral’s proprietary Advanced Thermal Control (ATC) system, a self-learning device that continuously monitors and adjusts to change in production parameters to achieve total process and product consistency. ATC has proven to reduce energy consumption by up to 20% with a 70% increase in stability. Extruders by design are sustainable machines with continuous processing and closely controlled parameters that enable extended operational lifetime. Screw and barrel wear are factors in feed production and most manufacturers offer premium metallurgy to extend time between replacements. Pretreating the ingredients in a preconditioner also greatly reduces screw and barrel wear. In addition, extruders allow complete production versatility. One system can easily make multiple products interchangeably and can be repurposed for different production lines when required.

Helping shrimp feed manufacturers reach sustainability goals As consumers search for sustainable food sources and nations seek to feed growing populations, farmed shrimp will likely face the same scrutiny as other farmed fish. By implementing sustainable practices, feed manufacturers can help shrimp farmers around the world to meet sustainability goals and produce highquality shrimp thus enabling all players to prosper from this growing market.

More information: Hadrien Delemazure Process Engineer and Feed Expert Clextral, France E: hdelemazure@clextral.com

LACTIC ACID BACTERIA FOR AQUACULTURE

Believe in what you see

We can see it inside, you will see it from the outside! BACTOCELL activates and associates with the gut mucosa, which is the key to a true probiotic effect. If you need to see more, years of research and field applications have provided compiling evidence of BACTOCELL’s modes of action and benefits at cellular, animal and farm level. Discover the world of BACTOCELL, the pioneering probiotic in aquaculture. Not all products are available in all markets nor all claims allowed in all regions.

LALLEMAND ANIMAL NUTRITION SPECIFIC FOR YOUR SUCCESS www.lallemandanimalnutrition.com

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High moisture extrusion: A promising new method for making aquafeed with unique properties Keshun Liu, USDA

Over the past several decades, extrusion processing has become the primary technique for making fish feed (Welker et al., 2019; Tacon, 2020). The method involves compressing a mixture of raw materials (a mash) through the barrel of a single or twin-screw extruder, applying heat to the mash as it passes through the barrel by rotating screw(s) and forcing the cooked mash through the small aperture of a die attached to the end of the extruder’s barrel. The whole process occurs at a high-temperature condition with a moisture content of the inner mash in the range of 15-45%. Upon exiting the die, the extrudate undergoes a sudden drop in pressure, causing rapid moisture loss and volume expansion. It is then cut into pellets and dried. The fish feed produced has a porous texture and acceptable durability. Starch (as in a grain flour) and/or other binder is often added to the raw mash at 5-20% to facilitate feed expansion and improve durability. However, the increased carbohydrate content is generally undesirable for carnivorous fish (Hemre et al., 2002). Compared with the prior technologies, such as expansion processing and steam pelleting, the high temperature and lowmoisture extrusion cooking has significantly improved both the physical and nutritional quality of feed (Welker et al., 2019). This popular method for making fish feed is commonly known as thermoplastic extrusion.

High moisture extrusion In parallel to the development of the low moisture extrusion technology for making aquafeed, over the past several decades, high temperature and high

Figure 1. Schematic diagram of a specially designed cooling die for the new extrusion method. Source: adapted from Liu et al. (2021).

moisture extrusion with a twin-screw extruder and inner mash moisture above 45% has been developed to impart a fibrous, meat-like texture to proteins of various sources in the food science community (Noguchi, 1989; Liu & Hsieh, 2008; Pietsch et al., 2019). The textured protein products can be used as meat analogs or meat extenders in a wide variety of ready-to-eat products. Therefore, in the food industry, high moisture extrusion has become an emerging technology for transforming vegetable proteins into palatable and consumeracceptable products. In contrast, in the aquafeed industry, there has been no single report on using high moisture extrusion to make aquafeed until recently when a pilot study was first published (Liu et al., 2021). According to the report, as part of a Trout-Grain Project, a prototype method

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Figure 2. Front view of an extruder in making the new high moisture feed. Source: Keshun Liu.

Figure 3. Cross-section views of conventional (left) and new (right) fish feed. Source: Keshun Liu.

of high moisture extrusion was developed at the U.S. Department of Agriculture research facilities in Idaho and Montana to make fish feed. The developing team consisted of Rick Barrows, a well-known fish scientist and now retired, Keshun Liu, a research food chemist with the USDA, and Jason Frost, a former USDA technician. The new process requires a twin-screw extruder, a uniquely designed cooling die (Fig. 1), and careful controls of extrusion parameters. During operation, a raw mash is mixed with water injected into the barrel, so that it has a moisture of 45-75%. The wet mash is then kneaded, heated and melted under high-moisture and high-temperature conditions, and then enters the specially designed die

attached at the end of the extruder. Within the die, the extruded mash is channeled into multiple elongated tubes, which are spaced so that cooling water can be circulated around within the outer house through the inlet and outlet connected to a water-circulating system. With this unique process, the hot extrudate is cooled immediately upon exiting the extruder’s barrel, while the pressure drop is controlled and gradual so that expansion of the extrudate is minimized (Fig. 2). Rapid cooling and controlled expansion prevent moisture loss and the formation of large air pockets within the extrudate.

High moisture extruded feed properties There are significant differences in physical properties between the high moisture extruded feed (HME feed) and a conventional feed made by the low moisture extrusion. The HME feed has a smaller diameter, denser structure and smoother surface (Fig. 3). When freshly made, the HME feed has an elastic feel and soft texture, with a hardness value approximately the same as that of a fish fillet. Upon contact with water, conventional feed (fresh or dried) rapidly disintegrates, while dried HME feed slowly returns to

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Figure 4. Changes in hardness of conventional and HME feeds with soaking time. (A) fresh feed. (B) dried feed. Solid circle: HME feed. Empty circle: conventional feed. Source: adapted from Liu et al. (2021).

the soft texture of its fresh stage, and both fresh and dried HME feed show resistance to breakage even after soaking in water for as long as 24 h (Fig. 4). Dry matter loss from the HME feed is significantly lower than the conventional feed after agitation in water for 6 h. Therefore, the HME feed has a softer texture, better pellet durability and significantly higher water stability than the conventional feed. Furthermore, the addition of a binding agent (such as wheat flour) becomes unnecessary when using the new method. Yet, the HME feed has some undesirable features as well. First, because of its denser structure, the HME feed has about 85% oil absorption capacity of the conventional feed. This makes it more difficult to produce floating and/or high-energy feed with this new method. Second, although the HME feed can be dried for easy handling and storage, its drying cost can be significantly higher than drying the conventional feed, due to the much higher moisture content of the former. In feeding aquatic animals such as fish, the interaction of feed with an aquatic environment poses problems not encountered in the feeding of terrestrial animals. This makes the physical properties of feed for aquatic animals more important than for terrestrial animals (Sorensen, 2012). Poor feed durability and water stability can negatively impact aquaculture profits and surrounding environments. In fact, the presence of lost nutrients and uneaten feed in fish farm effluent has been a major constraint for the expansion of

commercial aquaculture (FAO, 2017). From this perspective, the desirable features of the HME feed, which include soft texture, high pellet durability, high water stability, plus no requirement for a binder, can compensate for the undesirable feature of high drying cost and dense structure. Therefore, the new method of high moisture extrusion developed by the USDA scientists to make fish feed represents an emerging technology for the aquafeed industry with exciting implications. For example, it can be a better way to produce soft pellets that more closely resemble natural feed and thus are more attractive for Atlantic salmon and other species. More importantly, the new process can potentially revolutionize how feed is made and serve as a promising strategy to mitigate water pollution issues associated with aquaculture. Further research is needed for optimizing the new method and exploring its possible applications in making various types of aquafeed. References available on request. More information: Keshun Liu, Ph.D. Research Chemist National Small Grains and Potato Germplasm Research Unit USDA, Agricultural Research Service, USA E: Keshun.Liu@usda.gov

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Fish protein concentrate can partly replace water as plasticizer in the fish feed extrusion process Tor Andreas Samuelsen, Nofima

Consistent and high physical quality of feeds to aquatic species are crucial for efficient feed transportation, feeding logistics and reduced feed loss to the aqueous environment. Extrusion processing is a technology that enables the manufacturing of such quality and is the dominating technology used in commercial production. The extrusion process involves several processing steps to transform the dry recipe powder feed mix into expanded extrudates that can be dried to durable pellets and added oil in a vacuum coating operation. To obtain high physical pellet quality, the feed mix is moistened and heated by water and steam addition in the preconditioner and further heated by the use of mechanical mixing and viscous heat dissipation in the extruder barrel. During this processing, the powder is transformed into a plasticized and flowable material that can be shaped through the extruder dies. Normally a plasticizer in the form of water is added to reduce the temperature needed for this transformation and improve cooking efficiency in the extruder barrel. A plasticizer is a small molecule that penetrates the dry powdery feed mixture and weakens the intermolecular binding forces in the particles. The particle polymer chains then unfold and slide past each other creating a plasticized melt. The use of fishmeal in salmon feed formulations has been reduced from 65% to 15% in the last two decades and has partly been replaced by alternative plant-based ingredients, with soy protein concentrate (SPC) and vital wheat gluten as the dominating protein sources. Plant proteins, especially SPC, demand high water level and temperature in the extrusion process to obtain satisfactory plasticization and durable pellets. This gives

Figure 1. Capillary rheometer with pre-shearing capabilities. By setting the piston speed (shear rate) and measuring the pressure drop across the capillary die, melt viscosity can be calculated.

the feed manufacturing industry challenges related to pellet quality and drying costs. The use of alternative plasticizers to partly replace water can reduce extrudate moisture content and energy requirements in the dryer and improve physical feed quality. Our earlier studies have documented that the water-soluble peptides and amino acids in fishmeal have plasticizer effects comparable to water and improve physical pellet quality. We have also studied the plasticizing effect of the amino acid proline on SPC, and a near equal plasticizing effect for proline compared to water was found. This demonstrates that free amino acids and low molecular weight water-soluble peptides can be used as plasticizers and partly replace water in the extrusion process.

Fish protein concentrate Fish protein concentrate (FPC) is a hydrolysate produced by acid conservation and autolysis of

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Dalton) and >80% with molecular weight less than 500 Dalton, components with potential plasticizing effect. In industrial manufacturing practice, FPC may thereby represent a cost-effective and sustainable plasticizer solution.

Figure 2. Levels of FPC, water and SPC in the design.

fish offcuts and residues from filleting, gutting and other fish processing operations (silage process). It is a sustainable alternative protein ingredient. The raw material is minced and added formic acid to reduce the pH ≤ 4. This gives optimal conditions for protein hydrolysis by the fish digestive enzymes and a microbiological stable product. The hydrolyzed crude fish silage is heated and processed over a 3-phase decanter centrifuge to remove oil and particles before concentrated to FPC with a dry matter (DM) of 40-50%. A typical FPC can have a water-soluble protein content >90 g/kg DM where >60% is free amino acids (<200

Plasticizing effect of fish protein concentrate To document a possible plasticization effect of FPC, we performed a technical trial by using a capillary rheometer with pre-shearing capabilities (Fig. 1). This rheometer has been reported to give results comparable to the extrusion process and enabled us to produce a plasticized/melted feed mass in a conical shear cell prior to viscosity measurements through a capillary die and evaluation of dried extrudate physical properties. In the trial, we chose to vary the three ingredients, FPC, SPC and water in feed mixes. This was performed by use of a mixture design where the three ingredients always sum up to 100 g in a triangle (Fig. 2). The SPC and FPC have different fat and water content and we, therefore, varied the amount of SPC and FPC based on fat-free dry matter (FFDM). We held the rest of the feed mix constant (900 g) with a fixed composition consisting of fishmeal (100 g FFDM/kg), vital wheat gluten (100 g FFDM/kg), whole wheat flour (100 g FFDM/kg), SPC (354.7 g FFDM/kg), water (200 g/ kg; sum of moisture content in the ingredients + added water) and fat (45.3 g/kg; sum of fat content in the ingredients + added sunflower oil). Fat is a lubricator, and it was therefore set equal in all samples to prevent fat from affecting the results. We aimed to simulate the conditions at a preconditioner outlet for a “normal” salmon diet with a moisture content ranging from 20-30%, prior to simulating the extrusion process in the capillary rheometer.

Figure 3. CT-scan of extrudate samples for maximum concentrations of SPC, FPC and water and equal mixture of the three (centroid). Black is air, grey is the pellet structure, and white is bone fragments from fishmeal.

Results We operated the capillary rheometer at 110°C and a wall shear rate of 300 1/s, and

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Figure 4. Results for melt viscosity (Pa s), extrudate hardness (N) and density (g/L), and the extrudate microstructure parameter, open porosity (% of total porosity). The contours are flagged with the actual units. The red line represents feed mixes with similar values for hardness, density, and open porosity.

freeze-dried the extrudates into durable pellets. We thereafter measured pellet hardness by use of a texture analyzer, density, and microstructure by use of X-ray microtomography (CT-scan). The measured parameters showed high variation (Fig. 3 and 4) and with the physical pellet parameters hardness and density negatively correlated to the microstructure parameter, open porosity (i.e., % of total pore volume connected to the pellet surface).

Effect on melt viscosity We observed a decrease in melt viscosity when SPC was replaced with FPC and/or water but with a sharper decrease by using water (Fig. 4). This may be a result of the much lower viscosity of water compared to FPC and differences in how they interact with the other ingredients in the feed mix. We chose to hold the temperature, shear rate, and die diameter constant and the observed reduction in viscosity with increased concentration of FPC and/or water reflects a higher

effect of temperature on viscosity reduction in the feed melt phase. This confirms a plasticizing effect of the two ingredients.

Effect on hardness, density and open porosity We also observed that increasing the level of both FPC and water in the feed mixes increased pellet hardness and density and reduced the open porosity (Fig. 4). This may be a result of increased plasticization (cooking efficiency) and reduced melt viscosity, contributing to the formation of denser and harder pellet with a smoother outer surface as shown in Figure 3. From Figure 4, it can be seen that adding FPC had a higher effect compared to water. In contrast to water, which needs to be removed from the dryer, FPC will be carried over to the dried pellets. This will change the viscoelastic properties of the pellets and also give less pore structure, denser pellet, and increased hardness. We performed all the measurements under equal conditions in the capillary rheometer and as can be seen

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from Figure 4, SPC plasticized with FPC gave different extrudate properties compared to water plasticization. However, FPC added at a concentration of 65 to 70 g/kg FFDM at 20% moisture content gave values of hardness, density, and open porosity similar to SPC added 100 g/kg of water (30% moisture content; Fig. 4, red line). This documents that FPC can partly replace water as a plasticizer in the extrusion process, with a potential for a significant reduction of the energy consumption during drying of the wet extrudate.

Conclusion The pre-shearing capillary rheometer enables plasticization of the sample material prior to rheological and physical measurements relevant to extrusion cooking. The ratio between the ingredients SPC, FPC and water gave different melt viscosity, extrudate hardness, density and microstructure. At equal levels, SPC plasticized by FPC gave different extrudate properties compared to water, but by knowledgebased use, FPC can be a sustainable plasticizer and nutrient in extruded fish feed and partly replace water in the extrusion process. Acknowledgments This article is based on Ahmad, R., Oterhals, Å., Xue, Y., Skodvin, T., Samuelsen, T.A. (2019) Impact of fish protein concentrate on apparent viscosity and physical properties of soy protein concentrate subjected to thermomechanical treatment. Journal of Food Engineering, 259, 34-43. Laboratory and pilot equipment for technical ingredient characterization, feed production and product evaluation are now available at the new Aquafeed Technology Center hosted by Nofima (https://aquafeed.science/). References available on request.

More information: Tor Andreas Samuelsen Senior Scientist Nofima E: tor.a.samuelsen@nofima.no

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Pigmentation and growth performance on seabream and shrimp Didier Coulmier, Désialis and Nicolas Robert, Aquaculture Consultant Nutrition & Health

In the current competitive market of farmed seabream, fish quality differentiation is vital for producers. Among good conformation, homogenous weight, pigmentation of seabream is one of these quality criteria that makes a difference and is easily recognizable by the advised final customer. The name itself, “gilthead seabream” is a reminder of the importance of the face coloration of this animal in the wild. The hue of wild coloration is mainly yellow, gold yellow, with possible pink irisations. Moreover, in the 30 last years, aquaculture has grown a lot. As a result, the demand and price of raw materials, especially fish protein dedicated to the feed industry has increased dramatically. This trend leads producers to find alternative protein sources, often vegetable proteins. For years now, natural raw material has been used with success for pigmentation of fish and crustaceans: the extract of alfalfa named “PX Agro”, an exclusive product of Désialis obtained from a unique extraction process. From the alfalfa forage, an exclusively physical process, collects a juice by press, that is then coagulated, centrifuged and dried to obtain the alfalfa extract, also named alfalfa protein concentrate. PX Agro contains a high level of pigments called xanthophylls, mainly lutein and zeaxanthin, in an amount much higher than corn gluten meal for instance (3 to 4 times more). PX Agro is also rich in proteins, with an interesting amino acid profile for feed formulators. Its phosphorus level is adequate to fish requirements and is in a very available form. This product is available in crumbles or pellets for feed manufacturers and is incorporated as raw material in feed recipes for fish such as seabream but also with success in shrimp feeds.

We briefly relate the results of accurately run pilot tests in well-known research centers as IMBC for seabream.

Seabream coloration Tests show a very clear effect on seabream pigmentation compared to the control, after 7 weeks (temperature 19-22°C). Operculum shows a bright gold yellow area, as well as on the front area, a clear 1 to 2 cm yellow line is also visible on the low part of the abdomen. Seabream conformation We find in this seabream test a hepatosomatic index reduction (liver size compared to body weight) of

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Moreover, PX Agro incorporation in feed allows an FCR reduction of more than 6%, with improved growth of 6.5%, without change on the feed intake (self-feeder). PX Agro revealed much more efficiency than corn gluten meal for instance, on pigmentation as well as on growth performances.

Other species These growth performance improvements were also observed in other species on which PX Agro was used. In rainbow juvenile trout production, weight gain per unit energy intake was increased by 4% and in pacific shrimp, an animal that grows by molting process, weight gain was improved by 25-30%. This phenomenon can be explained by energy supply, and probably improved metabolism, osmotic balance, as well as supply of a rare essential component. In shrimp, PX Agro pigments are metabolized in astaxanthin, which leads to strong red coloration of shrimp shells after cooking. This metabolism is specific to crustaceans and does not occur in our farmed marine fishes.

Source: IMBC.

In a nutshell As a vegetable natural protein-rich in pigments, PX Agro, brings a “quality plus” for seabream pigmentation as for other aqua production, and improves growth performance and feed efficiency at the same time, which is welcome for producers. PX Agro has the strong advantage to be a natural, non-GMO complex raw material, combining pigments, proteins and other interesting nutrients such as vitamins, minerals and amino acids. References Robert N, Coulmier D, Chatzifotis S, Divanach P, Cuzon G. 2004. 11th International Symposium on Nutrition and Feeding of Fish, Pukhet Island, Thailand.

More information: Source: Ifremer.

around 6 to 8%, which would once again let us think of improved metabolism. This effect has also been observed on trout.

Didier Coulmier R&D Director Désialis, France E: didier.coulmier@desialis.com

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Recent trials show large potential for nutritionally improved feather meal in shrimp feeds Mélanie Guédon, Akiolis

Figure 1. New generation of feather meal.

Easily available on the market, being part of a sustainable production chain, and more economic than fishmeal, does feather meal have potential in shrimp farming? Still little used, it offers potential as an alternative to fishmeal. Akiolis, a French producer of ingredients based on poultry byproducts, has explored the use of this raw material in a series of trials, confirming that

feather meal can effectively replace a large part of fishmeal in shrimp feeds. Fishmeal is becoming scarce and the search for alternative proteins has already been at the heart of development projects of the aquaculture industry for several years. Among the raw materials allowing a supply of quality proteins to replace fishmeal, poultry meal has a proven track record. But this is

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Table 1. Feed formula tested.

Ingredient

Control Diet

Test Diet

Fishmeal LT, 70% CP

22 15

Feather meal

0 7

Wheat flour

33

35

Soybean meal

36.3

33.4

Fish oil (tuna oil)

3.0

3.0

Soy lecithin powder

1.5

1.8

Cholesterol

0.2

0.2

Mono sodium phosphate

0.5

0.5

Additives and micro-ingredients

3.5

4.1

Composition

Crude Protein

37%

37%

8%

8%

Crude Fat

not the only solution, other processed land animal proteins can fulfill this role, among them feather meal. Backed by its R&D center, Akiolis has developed innovative processes to guarantee a high protein digestibility of feather meal. After studying digestive performance and growth potential in fish species for many years, the recent focus has been on the use of nutritionally improved feather meals as fishmeal replacer in feeds for whiteleg shrimp. Global demand is strong and shrimp farming is moving towards a more technical and optimized model with an important parameter to the evaluation of alternative proteins in shrimp: resilience and immune sensitivity. Some aquafeed producers and farmers had concerns about feather meal and Akiolis’ nutritionally improved feather meal needed the best technical documentation. The company performed some trials in shrimp to reinforce the value of the company’s three feather meals, but beyond that, they have opened up opportunities for the entire shrimp industry and best-in-class feather meal producers and play a pioneering role, opening up avenues to overcome reluctance.

Promising prospects for the shrimp farming industry IMAQUA, a research company with roots in the University of Gent in Belgium, was contracted for this work. Their researchers quantified the effect of using feather meal to replace high-quality fishmeal in diets for

whiteleg shrimp (Penaeus vannamei). Previous studies in shrimp have often focussed on growth performance and feed efficiency. This was also done in this study. However, to prove the applicability of tested products to field conditions, shrimp were exposed to typical environmental stress (salinity change) and a pathogen challenge (WSSV) as part of the testing protocol. Moreover, histology observation of the hepatopancreas and immune gene evaluation were also conducted in order to get a full picture of the consequence of the change of diet in the metabolism of the animals.

A robust protocol that confirms the value of three types of qualitative feather meals The protocol was established with a reasonable ambition to replace 30% of fishmeal. A reference diet containing 22% of fishmeal was compared to three test diets, each one containing 15% fishmeal and 7% feather meal. The formulation has been controlled to guarantee an iso-proteic and iso-energetic value of each diet, as well as the balance of amino acids and micro-nutrients. Three different feather meals of Akiolis were tested: one produced with a classical process, the second one with a patented low-temperature drying (LT) and the third one with a new generation, innovative patented technology, this new feather meal is being launched this year (Table 1). Shrimp, with a start weight of 0.2 gram, were fed for 28 days at 27°C and 20 ppt salinity in four replicate tanks per treatment. After the feeding period, an average weight of 1.1 grams was achieved. Survival rate, feed conversion ratio (FCR) and growth rate (SGR) were not impacted by the inclusion of feather meal of all types. A well-balanced metabolism was documented through healthy histology of the hepatopancreas. Sections did not show any significant difference in the lipid droplets area. In further studies, the shrimp were subjected to a rapid decrease of salinity and challenged with inoculation of the white spot syndrome virus (WSSV). These tests were then supplemented by analyzing

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the gene expression of genes related to immunity. No difference was observed between any of the dietary treatments. Resistance to hypo-osmotic stress and WSSV-related mortality were identical in diets with a high level of fishmeal and diets containing feather meal. Translating such laboratory trials into a practical pond situation is often debated. However, the width of response criteria in this study does mimic many of the potential naturally occurring challenges to shrimp survival and performance, and thus these results document that feather meals from Akiolis can replace a significant share of fishmeal in shrimp feeds in practice.

These multi-criteria studies clearly demonstrate a high potential for using feather meals from Akiolis in shrimp feeds and confirm the company’s strategy to develop new products in the recently renamed Hydrofaks range, dedicated to aquaculture. The group is investing in new drying and granulation technologies that increase the nutritional value, stability and digestibility of its proteinbased products. As a key actor of the circular economy, Akiolis provides sustainable, healthy and traceable ingredients to support the success of the fish and shrimp farming industry. References available on request.

Conclusions These tests indicate that we can reduce dependence on wild resources to feed farmed shrimp, by reducing the rate of fishmeal in their ration from 22% to 15%, without impacting productivity. They thus show that this substitution of fishmeal by feather meal is possible on a 1 to 1 basis, and that feather meal can be included up to 7% - as long as it has all the right quality characteristics, such as those tested here. These results offer new formulation perspectives for aquafeed producers.

More information: Mélanie Guédon Product & Market Manager Akiolis, France E: melanie.guedon@akiolis.com

PIGMENTATION AND GROWTH PIGMENTATION AND GROWTH PERFORMANCE PERFORMANCE 11 100 % natural xanthophylls pigments 100 % natural xanthophylls pigments for greater golden or orange-pink coloring

for greater golden or orange-pink coloring for for feeding feeding fish fish and and crustacean crustacean

22

Pellets or crumble Pellets or crumble

High High quality quality vegetable vegetable protein protein content content for for improved improved growth performance growth performance

3

French origin, non GM product, «VLOG geprüft»

DESIALIS - 12 rue de Ponthieu 75008 PARIS - + 33 (0)1 42 99 01 01- www.desialis.com - info@desialis.com

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All-natural: Immune protection, enhanced performance Christian Cordts, Mirona Quandt, Berg + Schmidt

In the fight against bacteria and viruses, there are high hopes for the role played by medium-chain functional lipids. Functional vegetable lipids can improve gut health by positively affecting the microbiota and immune function. Believe it or not, the antimicrobial properties of medium-chain functional lipids have been known since the 1800s, but have previously been overlooked as a value-added feed additive due to the use of antibiotics. Functional lipids incorporated into fish feeds are seen as a method to support immune function, resistance to stress and pathogens, and increase fish production. As such, German feed ingredients manufacturer Berg + Schmidt offers natural functional lipids that support the immune system and growth of fish. Thanks to expert knowledge and innovative technology, the company’s functional lipids and fats are easy to use and offer numerous health benefits.

Gut health: Epicenter of immunity The hot topic of gut health encompasses a number of factors. These range from an effective digestive function, optimal absorption of nutrients, the absence of gastrointestinal diseases, good immune status, healthy gut flora to the wellbeing of fish and shrimp. The gut microbiota, which resides in the gastrointestinal tract, provides essential health benefits to its host, particularly by regulating immune homeostasis. Moreover, it has become obvious that alterations to the gut microbial composition can cause immune dysregulation. To maintain this delicate status, a balance between nutrient intake, symbiotic microflora and an optimized mucosa must be maintained. Any negative impact on gut flora, e.g. by oral administration of antibiotics, stressful husbandry conditions, an unbalanced diet or stress, can result in reduced

Figure 1. Major points for a healthy gastrointestinal system.

functionality and immune power of the gut. Here, medium-chain functional lipids play a key supporting role.

Smart way to beat pathogens The antimicrobial properties of fatty acids have been known and used for many decades. However, antibiotic-resistant bacteria, legal regulations and increasing consumer demand for sustainable solutions have driven a need for alternatives such as those found in functional lipids. Medium-chain fatty acids, monoglycerides and triglycerides are saturated fatty acids with carbon lengths of 6, 8, 10 and 12, named caproic, caprylic, capric and lauric acids, respectively. Due to their chain length and unique structure (which is shown in Fig. 2) they can be used as antimicrobial, antiviral, antifungal and antiinflammatory agents, and can be referred to as medium-chain functional lipids.

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In nature, these lipids are found in palm kernel, coconut oil and milk fat. The most effective and powerful form of medium-chain functional lipids is achieved by esterifying with glycerine to form a monoglyceride. It is vital to attach the fatty acid in the 1-alpha position to achieve maximum efficacy of the medium-chain monoglyceride. This chemical structure provides fatty acids with high emulsifying properties, which enables them to interact with both water and lipids. Typically, free fatty acids exhibit their greatest emulsifying properties at lower pH in their non-ionized form. However, monoglycerides do not have a carboxyl group but instead are bound to glycerol. This imparts monoglycerides with emulsifying properties without being pH sensitive. Depending on the temperature, they can be liquid or solid for use in a wide range of applications. The antimicrobial mode of action of medium-chain functional lipids is believed to fall into different general categories: increased cell permeability, inhibition of enzymes and nutrient uptake, and disruption of the electron transport chain. Medium-chain functional lipids form double-layered micelles that can fuse with the bacterial cell membrane. This can result in the formation of membrane surface pores and tubes, causing cell leakage. At high enough concentrations,

fatty acids can completely solubilize membrane structure and fluidity, impairing the receptors and enzymes necessary to import key nutrients. To disrupt the electron transport chain in the cellular membrane, they bind directly to the transport carriers. This changes the structure via increased membrane fluidity, resulting in less energy for the cell. Other proposed modes of action for medium-chain functional lipids are the lowering of pH inside and outside the cell, reduction of pathogen toxin production, interference with pathogen adhesion and disruption of cell-to-cell signalling.

Trials with shrimp and fish A study with Penaeus vannamei shrimp, conducted at the Kasetsart University (Bangkok, Thailand), demonstrated that LipoVital Protect Aqua, a special combination of medium-chain fatty monoglycerides, could improve performance and support gut health of fish and shrimps. The trial focused on growth, survival, immune response and intestinal bacteria composition. Numerous preliminary studies in vitro and in vivo compared the synergistic effects of monoglycerides against specific aquaculture pathogens. To determine the effects of LipoVital Protect Aqua on the resilience of white shrimp, the animals were especially challenged with low dissolved oxygen in laboratory conditions.

Figure 2. Structure of medium-chain fatty acids.

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Figure 3. Application of the monoglyceride form of Lipovital Protect Aqua.

In conclusion, the study proved that a naturally sourced and specifically composed mix of different medium-chain functional lipids, increased immune function and performance, and reduced the mortality rate. Moreover, LipoVital Protect Aqua reduced the count of Vibrio spp. Figure 3 shows a dosagedependent reduction in the total Vibrio count. Increasing the length of the feeding period with medium-chain functional lipids showed a further reduction in potentially pathogenic bacteria. At farm level, LipoVital Protect Aqua contributes to more profitable and sustainable production. LipoVital can be supplied as a high-purity raw material, and as a customized compound with desired physical criteria. LipoVital is effective at low doses. The inclusion rate can be increased under challenging conditions. Berg + Schmidt is currently working with renowned institutes on further trials for the use of LipoVital in aquaculture, with promising initial results in salmon and shrimp feeding.

Lecithins: Another powerful ingredient Besides monoglycerides, lecithins are also a powerful additive providing essential nutrients, stimulating the metabolism, strengthening the immune system and providing high energy content. Furthermore, lecithins are emulsifiers and thus play an important role in fat digestion, and also enable the feed producer to unlock the full energy potential of added oils and fats. Obtained from plants such as soy, rapeseed or sunflower, lecithins can be processed into powders

or liquids. Added to feed solutions for aquaculture, they boost the growth rate and development of young fish, advance the feed conversion and liver metabolism. As an ingredient with added value, lecithins can also facilitate production processes. Their excellent emulsifying capacity improves the miscibility of raw materials and gives pellets or flakes greater elasticity. In addition, production throughput is increased because lecithin acts as a lubricant. Thanks to their antioxidative qualities, finished products score with a longer shelf life. Since the 2000s, Berg + Schmidt has supplied proven products to the aquaculture business.

More information: Christian Cordts Product Manager Feed Additives Berg + Schmidt GmbH & Co. KG

Mirona Quandt Product Manager Feed Additives Berg + Schmidt GmbH & Co. KG E: feed@berg-schmidt.com

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Nutritional fish & shrimp pathology - II Albert G. Tacon, Ph.D. Dr. Albert Tacon is a Technical Editor at Aquafeed.com and an independent aquaculture feed consultant. E: agjtacon@aquahana.com

Disorders in lipid and fatty acid nutrition This column is the second of a six-part series extracted from a forthcoming publication by the author dealing with the major reported nutritional disorders in farmed fish and shrimp, and represents an update to a previous review published by FAO in 1992 (Tacon, 1992)1.

Dietary essential fatty acid deficiency Dietary essential fatty acids (EFA) are important components of fats and oils and may include (depending upon the fish or shrimp species): linoleic acid (18:2n-6), linolenic acid (18:3n-3), eicosapentaenoic acid (EPA 20:5n-3), docosahexaenoic acid (DHA 22:6n-3), and/or arachidonic acid (20:4n6). Dietary EFA are defined as those fatty acids which the fish or shrimp species in question are incapable of synthesizing at a sufficient rate to meet metabolic demand. EFA have numerous important biological roles within the animal body, including serving as essential structural components of all cell membranes, intracellular signaling, hormone production and energy production. Table 1 shows the major reported deficiency signs and health impacts of fish and shrimp fed EFA deficient diets. As with individual amino acids, the EFA also have functional properties, and as such apart from a basic dietary requirement to prevent classic nutritional pathologies for optimum growth and feed efficiency, there is also an additional possible requirement for maximum growth and optimum health under stressful environmental conditions. Oxidization of dietary lipids Dietary lipids (including phospholipids, EFA, cholesterol, fat soluble vitamins, and carotenoids) are highly prone

to oxidative damage and degradation on prolonged storage; degradation usually being by autooxidation and combing with oxygen via free radical reactions and the subsequent formation of toxic oxidation products, including lipid hydroperoxides and secondary breakdown products. Table 2 shows the reported deleterious effects of feeding oxidized lipids to fish and crustaceans. It is important to mention, that the deleterious effects of oxidized lipids are two fold, namely 1) the direct toxic effects of the primary and secondary lipid degradation products (hydroperoxides, conjugated dienes/trienes, carbonyls, aldehydes, volatiles, malondialdehyde, oxysterols, etc.) and 2) the loss of essential nutrients through the oxidation process such as the highly reactive long-chain polyunsaturated fatty acids (EPA, DHA) and tissue antioxidants, including a-tocopherol (vitamin E), vitamin C, carotenoids and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase). Notwithstanding the above, under practical farming conditions, the potential deleterious effects of oxidized or rancid dietary lipids can be reduced and/ or eliminated through the increased use of dietary a-tocopherol, and to a lesser extent with supplemental vitamin C, vitamin A, selenium, vitamin premix, and glutathione. In addition to the use of natural antioxidants, it is also common practice for feed ingredient suppliers and feed manufacturers to use synthetic antioxidants to prevent lipid peroxidation, including ethoxyquin, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and propyl gallate (PG). Although the efficacy of synthetic antioxidants is generally less than a-tocopherol, their ready availability

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Table 1. Reported essential fatty acid (EFA) deficiency signs and health impacts in farmed fish and shrimp.

Major species – EFA

Reported EFA deficiency signs & health impacts 2

Atlantic salmon (Salmo salar) Increased fry mortality, increased heart weight and atheriosclerotic alterations, altered gut microbiota

Blue shrimp (Penaeus stylirostris)

Reduced resistance to environmental stressors & immune response

Blunt snout bream (Megalobrama amblycephala)

Reduced immune function and antioxidant status

Chum salmon (Oncorhynchus keta)

Increased mortality, swollen pale livers

Dover sole (Solea solea)

Reduced larval tolerance to hypoxia

European sea bass (Dicentrarchus labrax)

Reduced non-specific immune function

Gilthead sea bream (Sparus aurata) Reduced erythrocyte volume, increased erythrocyte fragility, hemoglobin count, red blood cell count, and reduced cellular immunity, reduced egg production and egg quality, liver granulomatosis, extensive hemorrhages of internal organs, liver degeneration, pericarditis and endocarditis, high occurrence of hydrops in larvae Grass carp (Ctenopharyngodon Idella)

Vertebral column curvature (lordosis)

Greasy grouper (Epinephelus tauvina & E. fuscoguttatus)

Larvae – shock syndrome

Japanese seabass (Lateolabrax japonicus)

Reduced immune response

Milkfish (Chanos chanos)

Increased eye abnormality in larvae, increased larval mortality

Nile tilapia (Oreochromis niloticus)

Swollen pale livers and high liver lipid content

Palmetto bass (Morone saxatilis x M. chrysops)

Shock syndrome – larvae

Rainbow trout (Oncorynchus mykiss) Reduced reproductive performance and egg quality, including reduced eyed eggs and hatchability, fin erosion, heart myopathy, shock syndrome, Increased mortality and shock syndrome, poor appetite, shock syndrome, reduced immune response, histological alterations Red drum (Sciaenops ocellatus)

Increased mortality, fin erosion, shock syndrome

Striped Jack (Pseudocaranx dentex)

Increased mortality

Turbot (Scophthalmus maximus) Altered gill structure, disappearance of chloride cells, "sloughing off" of epithelium along the primary and secondary filaments, accumulation of cellular material in inter-lamellar spaces White fish (Coregonus lavaretus maraena)

Swollen pale livers with histological alterations

White shrimp (Litopenaeus vannamei)

Reduced antioxidant enzyme activity

Yellowtail (Seriola quinqueradiata) Increased larval mortality (hydrops – floating bodies), disrupted schooling behavior 2

Major reported deficiency signs and health impacts in addition to reduced growth and feed efficiency.

and lower cost has meant that most lipid-rich feed ingredients sources (fishmeal, fish oil, terrestrial animal byproduct meals and fats) and finished aquaculture feeds, are usually stabilized with synthetic antioxidants, and in particular ethoxyquin. However, due to increasing human food safety concerns regarding the possible carry-over effects of synthetic antioxidant residues into fish and shrimp flesh, there is a growing trend toward the reduced use of synthetic antioxidants and the identification and use of more environmentally friendly natural antioxidant sources.

Other reported factors known to promote dietary lipid oxidation include 1) direct exposure of feed containing lipids to light (through photo-oxidation), 2) the presence of dietary pro-oxidants such as inorganic trace elements (such as iron and copper), and 3) the presence of feed ingredients containing lipoxygenase enzymes, such as lipase within rice bran. Tacon, A.G.J. (1992). Nutritional fish pathology: morphological signs of nutrient deficiency and toxicity in farmed fish. FAO Technical Paper No. 330, FAO, Rome.75 pp. 1

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Table 2. Reported deleterious effects of feeding oxidized lipids and diets to fish and shrimp.

Major species – EFA

Reported pathology and health impacts

Arctic charr (Salvelinus alpinus)

Reduced growth and higher hepatosomatic index

Atlantic cod (Gadus morhua) Increased erythrocyte osmotic fragility, decreased liver alpha-tocopherol and increased oxidative stress Atlantic halibut (Hippoglossus hippoglossus) Increased skeletal deformity: scoliosis and lordosis, reduced growth, reduced plasma glucose levels Atlantic salmon (Salmo salar) Reduced oxidative stress, lipoid liver degeneration (LLD) and accumulation of ceroid in hepatocytes Black sea bream (Acanthopagrus schlegeli)

Reduced growth and survival rate, increased hepatosomatic index

Channel catfish (Ictalurus punctatus) Poor growth, feed efficiency and survival; exudative diathesis, muscular dystrophy and depigmentation; fatty liver and anemia, reduced lipid metabolism and fatty acid synthesis, reduced growth, liver & kidney histological alterations, increased lipid droplet accumulation in the hepatocytes, mitochondrial vacuolation in the renal tubules Chinese longsnout catfish (Leiocassis longirostris)

Lighter tail skin coloration

Coho salmon (Oncorhynchus kisutch)

Reduced growth and feed efficiency

Common carp (Cyprinus carpio) Reduced growth, high mortality and muscular dystrophy or "Sekoke" disease, muscular dystrophy, damaged gut mucosa European seabass (Dicentrachus labrax)

Increased erythrocyte fragility and innate immune response

Grass carp (Ctenopharyngodon Idella) Severe intestinal mucosa damage, fatty liver, bile acid accumulation, increased oxidative stress and decreased antioxidant capacity of hepatopancreas, reduced intestine bile acid content, enhanced number of goblet cells, enlarged microvilli and gap between tight junction, hyperplasia and edema of villi, leading to damaged intestinal epithelial tight junction and destruction of the intestinal mucosal epithelium, and increased intestinal oxidative stress Hybrid catfish (Clarias macrocephalus x C. gariepinus) Jaundice disease, high mortality, yellow skin & gill pigmentation, enlarged spleen, kidney & gall bladder, pale yellow spleen, kidney, liver & body fat, with yellow ascitic fluid in their abdomen; histologically heavy deposits of hemosiderin and ceroid in the spleen, kidney and liver; decreased haematocrit, RBC count, hemoglobin content, hemolytic anemia associated with lipoid liver degeneration – these symptoms were believed to be due to the feeding of rancid chicken viscera Largemouth bass (Micropterus salmoide) Reduced growth and feed efficiency, increased oxidative damage, decreased hepatosomatic index, increased oxidative stress, histological alterations of liver with hepatocytes with lipid vacuoles and nuclear migration, severe oxidative damage and loss of reducing capacity Nile tilapia (Oreochromis niloticus) Marked congestion, with some hemorrhage, in dermal vessels around snout and at bases of pectoral/dorsal fins, lordosis, exophthalmia, abdominal swelling, cataract, orbital collapse, darkening of liver, marked distension of bile duct, steatitis of all abdominal fat bearing tissue, deposits of intracellular ceroid in liver, spleen, kidney and choroid, increased mortality Rainbow trout (Oncorynchus mykiss) Reduced growth & increased oxidative stress, lipoid liver degeneration due to feeds containing rancid lipids, reduced growth, microcytic anemia & liver lipoid degeneration, reduced growth & feed efficiency, severe muscle damage, increased mortality & increased erythrocyte fragility, reduced red blood cell numbers, hemoglobin content, hematocrit & increased hemolysis, increased mortality, rainbow trout fry syndrome (RTFS) - dystrophic changes in the liver, kidney and muscle, reduced growth & increased oxidative stress Red sea bream (Pagrus major)

Increased oxidative stress, reduced growth and increased oxidative stress

Rohu (Labeo rohita)

Reduced growth and increased oxidative stress

Senegalese sole (Solea senegalensis)

Reduced bone mineralization and skeletogenesis

Siberian sturgeon (Acipenser baeri) Reduced growth, survival, increased oxidative stress & larval deformity in the absence of vitamin A Turbot (Scophthalmus maximus)

Muscular dystrophy, reduced growth, reduced disease resistance

White shrimp (Litopenaeus vannamei)

Reduced growth, increased hepatosomatic index & oxidative stress

Yellowtail (Seriola quinqueradiata)

Reduced growth & hematocrit, high mortality

Yellow catfish (Pelteobagrus fulvidraco)

Reduced intestinal health & increased goblet cells in foregut

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How to enhance growth performance of tilapia Iris Kröger, Dr. Eckel Animal Nutrition

Performance-limiting effects in intensive aquaculture In intensive aquaculture systems with high stocking density, tilapia is exposed to environmental stress such as hypoxic stress and increased loads of pathogens. These challenges trigger a series of reactions of the immune system and are associated with a high risk of oxidative and inflammatory stress. Because of the high energy demand of immune cells, inflammatory reactions limit the growth performance of tilapia. Using flavonoids to boost health and performance In addition to management measures to reduce the risk of environmental stress, innovative feeding strategies hold great potential. In fact, phytogenic feed

additives can improve fish tolerance to environmental and inflammatory stress and thus enhance growth performance. For example, dietary plant flavonoids can reduce inflammatory responses in the cells and strengthen them against hypoxic stress (Xia et al., 2020). In addition, their antimicrobial and anti-inflammatory properties have been repeatedly demonstrated (Sae-Law et al., 2017). Furthermore, their properties to increase weight gain in fish, for instance by feeding green tea extracts and quercetin, have been highlighted (Zhai & Liu, 2013; Chandravanshi et al., 2020). This shows the potential offered by single flavonoid sources to improve health and performance in aquaculture.

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Figure 1. Effect of Anta®Ox Aqua on growth performance of tilapia.

Since different flavonoids unfold different effects in vivo and in vitro, optimal performance, for example in tilapia, is likely to be achieved by combining different flavonoid sources. As recently demonstrated, a combination of selected flavonoids significantly enhanced the performance of farm animals and shrimp (Gessner et al., 2008; Niyamosatha et al., 2015; Shata et al., 2019). These results suggest that the feed additive Anta®Ox Aqua, with its high content of flavonoids from grapes, green tea and hops, may contribute to the enhancement of growth performance in tilapia.

Investigating growth-promoting effects of Anta®Ox Aqua in tilapia Based on the promising findings of the effects of flavonoids in aquaculture (Zhai & Liu, 2013; Niyamosatha et al., 2015; Chandravanshi et al., 2020), we conducted a trial to investigate growth-promoting effects of Anta®Ox Aqua (AOA). This feed additive contains a carefully assembled formulation of different flavonoid sources to exert synergistic effects and is characterized by small particle size, optimizing bioavailability in the animals. We hypothesized that the effects of AOA would be more potent than those reported by Chandravanshi et al., 2020 and Zhai & Liu, 2013. During the experiment, 342 tilapia were kept in six tanks for eight weeks. The initial size of the fish was 1 cm with 0.1 g average body weight and a stocking density of 0.125 kg/m3. During the experiment, fish

were divided into two groups. While the negative control was fed commercial starter feed without any feed additive, the treatment group was fed the flavonoid-rich feed additive AOA (Dr. Eckel Animal Nutrition GmbH & Co KG) at a dosage of 0.5 kg/t of feed. Fish were fed manually three times per day. To determine the effects of AOA on growth performance, body weight and body length were measured individually.

Substantial effects on body weight and length Results showed that AOA increased the body length of tilapia by 9%. In addition, fish fed AOA for eight weeks had a 20% higher body weight than the fish in the control group (Fig. 1). This confirms the great potential of AOA for performance optimization in tilapia farming. The growth-promoting effects can in all likelihood be attributed to the synergistic combination of flavonoids in AOA, as flavonoids have been shown to increase immune capacity and reduce antioxidant and antiinflammatory effects in fish (Chakraborty, 2013; Zhai & Liu, 2013). The growth-enhancing effects of AOA that we observed were stronger than those in the study by Zhai & Liu, 2013. Here, the data reported that the plant flavonoid quercetin increased the body weight of tilapia by 12%. In their study, fish received a much higher dosage of the flavonoid, namely 1.6 kg quercetin/t of feed (Zhai & Liu, 2013). This indicates that AOA is more effective than feeding quercetin, even at significantly

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lower doses. As previously mentioned, this higher efficacy is most likely due to the combination of different flavonoid sources in AOA, which, in synergistic action, have been found to increase growth-promoting effects in tilapia. The next step was to compare the results of green tea to AOA. According to the authors of the study, the inclusion of green tea fed at a concentration of 0.5 kg/t feed enhanced the weight gain of carps by 15% (Chandravanshi et al., 2020). In this experiment, fish received the green tea for a period of 12 weeks. However, the growth-promoting effect at the end of the study was smaller than the effects of AOA in our study, which covered only an eight-week period. This suggests that AOA is already more effective than the administration of flavonoids from green tea in shorter periods of time. This higher effectiveness can, with reasonable certainty, be attributed to the selected combination of different flavonoid sources in AOA. Another explanation for the stronger effects observed in our study is the exceptionally small particle size. If phytogenics are present in very small particle size, this increases the specific surface area of the plant materials and thus the bioaccessibility and bioavailability of the flavonoids (Wang et al. 2014).

Conclusions Limited growth due to environmental and inflammatory stress is a serious threat causing severe economic losses in tilapia farming. Anta®Ox Aqua, a natural feed additive with a particularly small particle size containing a unique combination of different flavonoids, showed substantial growth-promoting effects in tilapia. Therefore, feeding concepts containing Anta®Ox Aqua are a promising means to optimize performance and improve profitability in tilapia farming.

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References available on request.

More information: Iris Kröger Technical Sales Manager Dr. Eckel Animal Nutrition, Germany E: i.kroeger@dr-eckel.de

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Hydronix-Aquafeed advert-April& 2021 58.4x228.6mm.indd 16:01 Aquafeed: Advances in Processing Formulation Vol11326/03/2021 Issue 2 2021


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Plant extracts in aquafeeds: Standardization as a key parameter Mathilde Buffiere, Nor-Feed, Rôger Oliveira e Silva, Federal University of Vale do São Francisco

Betting on each batch of a plant-based product “Plant extract” is a generic name of a very diverse group of compounds. From a semantic point of view, a plant extract is nothing but the product obtained after contact between a plant and a solvent. This process leads to the solubilization of some molecules from the plant into the solvent. The final product depends on many parameters, such as the part of the plant used (leaves, bark, roots, fruits, seeds, etc.), the kind of solvent (polar/apolar), the process (duration, temperature, pressure), etc. Even in controlled conditions, plant extracts obtained from two different batches can be very different. This is linked to the plant’s growing conditions or the so-called “terroir”, well known by wine lovers.

Secondary metabolites produced by the plants depends on their growing conditions, such as weather conditions, substrate composition and pathogen pressure. In the aquafeed industry, this “terroir” effect is not desired, as we are looking for standardized products. Standardization is then a prerequisite as it enables regular performance.

The compounds to standardize Quality Control Plan (QCP) is commonly applied to raw materials. Hence, feed formulators understand that nutrients such as protein, fat and ash are different from batch to batch and must be consequently and regularly analyzed to formulate an efficient feed. It is exactly the same for plant extracts composition. Let’s take the citrus extracts example. Citrus are well

Figure 1. Lemon extract thin layer chromatography clearly shows the difference between primary metabolites (osidic compounds) and secondary metabolites (active compounds).

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that they have prebiotic activity. They stimulate the growth of beneficial bacteria and inhibit the adhesion of pathogenic bacteria. They also stimulate SCFA production. For those reasons, Nor-Feed standardizes the POS content in every batch of Nor-Spice AB® that comes out of its production lines.

Standardizing efficiency in plant extracts Apart from analytical compounds, plant extracts can also be standardized for their activity. It means it’s possible to analyze the in vitro activity of the product for a given property. Nor-Spice AB®’s ability to stimulate the growth of beneficial bacteria is one of its QCPs. Indeed, this activity is checked in vitro for each and every production batch. The product can only be sold if it increases lactic Figure 2. Weight gain in 60 days and FCR in a trial with NOR-SPICE AB in tilapia. acid bacteria growth 7 fold or more compared to the same culture known for their positive effects on aquaculture species media without the product. The stability of the abovegrowth. Fine characterization of the commercial mentioned components allows customers to purchase product Nor-Spice AB® allowed the identification of a product whose effectiveness is constant, batch about 40 secondary metabolites, including after batch. 17 citroflavonoids such as eriocitrin, hesperidin Standardization, extrusion and plant protein and naringin. Some of these molecules are well The efforts made by the Nor-Feed R&D team in described in scientific literature. Their types of collaboration with FeedInTech Laboratory to develop action encompass beneficial bacteria growth precise, repeatable and efficient analytical methods stimulation, inhibitory effect on several bacterial are not only useful to characterize the additive itself species and short-chain fatty acids (SCFA) production but also to analyze product behavior during the feed stimulation. For these reasons, Nor-Feed chose manufacturing process. As a plant extract is basically to standardize citroflavonoids in the product. made of plants, it is a particularly tricky task to isolate Apart from flavonoids, citrus are among the and analyze it in even more animal protein devoid most pectin-rich fruits known to date. Pectins are aquafeeds. High-fat levels contained in fish feed complex polysaccharides known for their important might also interact with the analytical process. role in plant cell adhesion and maintenance, Thanks to in-depth R&D efforts, Nor-Feed team was allowing these cells to fuse with each other. able to develop a method to find and dose Nor-Spice Pectin hydrolysis leads to the production of pectic AB® in complete feeds made of plant products, even oligosaccharides (POS). Their influence on microbiota when the product is used with a concentration as low is well documented in the scientific literature. as 150 ppm. This development was also used to test Among other properties, it has been demonstrated ®

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the ability of the product to cope with the extrusion process applied in our partners’ plants.

of juvenile tilapia. They are the most recent example underlining the link between the use of a standardized product in active compounds and its effects on aquatic animal growth.

From the Petri dish to the pond The latest results obtained with Nor-Spice AB® in aquaculture species were gathered in Brazilian waters. The Aquaculture Laboratory team of the Federal University of Vale do São Francisco held a trial with Nile tilapia juveniles (mean initial weight = 12.8 ± 0.17 g). After a two-week acclimatization period, fish were divided into 5 groups containing 4 replicates each, where each group received a specific diet. The performance of the control group was compared with that of the animals whose diet was enriched with different doses of Nor-Spice AB®: 200 ppm, 400 ppm, 800 ppm, 1,600 ppm. The fish were fed for 60 days. Daily feed intake was measured for all tanks and each fish was weighed individually at the end of the trial. The data were then subjected to an ANOVA. Results demonstrate Nor-Spice AB®’s ability to positively influence the growth and feed conversion

References available on request.

More information: Mathilde Buffiere Citrozest Range Product Manager Nor-Feed, France E: mathilde.buffiere@norfeed.net

Rôger Oliveira e Silva Researcher Aquaculture Laboratory UNIVASF, Petrolina-PE, Brasil

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Phytogenic feed additives to counteract mycotoxin impact on fish health Fernando J. Sutili, ELOAQUA Consulting

Phytogenic feed additives Phytogenic feed additives (PFAs), also known as phytobiotics or botanicals, are plant-derived natural bioactive compounds which may be incorporated into animal feeds for purposes of improving health and welfare. Being derived from herbs, spices and other plants, PFAs are commercialized as extracts, essential oils, dried powder and isolated compounds as well as mixtures of these forms. In fish production, PFAs may be used as food flavorings, antimicrobials, immunomodulators, antioxidants, digestive stimulants and technological additives to enhance feed quality and safety. One major advantage of PFAs lies in the fact that they are generally safe and do not trigger the side effects often associated

with synthetic chemical utilization. In addition, they are cost-effective and environmentally friendly. Such properties and advantages make PFAs excellent growth promoters to be used in farmed fish.

Antifungal properties Even though bacteria and viruses are considered more important animal pathogens than fungi, these have a major relevance regarding pathogens on plants. In agricultural production, plant-pathogenic fungi are the major cause of yield losses. Throughout evolution, plants have developed a wide variety of defense mechanisms to protect against fungal attack. In nature, plants synthesize a vast array of phytochemicals which play a role in protection strategies. By exerting

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Table 1. Observed benefits of including phytogenic feed additive (PFAs) in aflatoxin B1 (AFB1)-contaminated fish diets.

PFAs Fish species Rosemary Oreochromis niloticus

Benefits Reduced the deleterious effects of AFB1 on growth, innate immunity and antioxidant activity.

Fennel essential oil O. niloticus

Mitigated the impact of AFB1 on immunological and antioxidant responses; decreased AFB1 residues in musculature and liver.

Black pepper O. niloticus

Reduced growth impairment caused by AFB1; lowered AFB1 residues in whole fish body.

Rosemary and thyme powder Cyprinus carpio

Restored digestive enzymes activity to near normal level.

Thyme essential oil Oncorhynchus mykiss

Decreased AFB1-induced growth and innate immunity suppression.

Tea tree oil

Rhamdia quelen

antimicrobial activity, these constituents chemically interfere with the synthesis or function of vital components of plant pathogens. As a result, both growth and toxin production of these microorganisms are reduced. Such natural antimicrobial properties make plantderived natural bioactive compounds a great biotechnological tool for the inhibition of fungi growth and, consequently, prevention of mycotoxins production during plant development in the field, storage and food or feed production. In fact, it is well documented that many types of essential oils, plant extracts and isolated compounds obtained from multiple plants and herbs exhibit intense antifungal properties. Furthermore, the use of PFAs as antimicrobials is associated with a low risk of resistance development.

Antioxidant properties Stressful situations for plants, like fungal attack, may trigger the production of reactive oxygen species (ROS), which activate the reactive antioxidant enzymatic and non-enzymatic responses. The nonenzymatic response is related to the synthesis of several phytochemicals (carotenoids, phenolic acids, flavonoids and others). Likewise, mycotoxins induce oxidative stress via ROS generation, stimulate inflammatory reactions, and induce molecular and

Reduced liver damage; enhanced antioxidant status.

cellular lesions in animals. An imbalance between ROS production and the antioxidant defense systems may cause DNA damage, lipid peroxidation, protein damage and cell death. When added to feed, natural antioxidants synthesized by plants have been shown to mitigate and/or prevent the health burden of mycotoxin exposure on animals. Antioxidants have also been added to commercial feeds to prevent lipid peroxidation and oxidative rancidity during production, processing and storage. Exogenous antioxidants help to preserve the sensory qualities of feed and to prevent the destruction of critical nutrients as pigments and vitamins. The feed/ food industry has become increasingly interested in natural antioxidants because some synthetic antioxidants have shown potential animal and human health hazards. When directly applied to feeding stuff, plant-derived bioactive compounds may therefore improve product shelf life and safety.

Detoxification properties Several approaches can be taken to minimize mycotoxin contamination in animal feeds, including prevention of fungal growth and mycotoxin formation. Another strategy used to reduce or eliminate mycotoxins from contaminated commodities is the inclusion of feed additives in animal diets. Detoxification seems to be the most attractive way to address the mycotoxin problem.

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It may involve physical, chemical or microbiological processes that are applied to detoxify by destroying, modifying or absorbing the toxin. Nonetheless, every treatment has limitations. The most well-known approach to reduce mycotoxin exposure is to decrease their bioavailability by adding mycotoxin-adsorbing agents to animal feed. Such compounds are able to bind and immobilize mycotoxins in the gastrointestinal tract, thus lowering bioavailability. Despite eliminating the risk of certain mycotoxins successfully, this procedure is not effective for all major mycotoxins in feed commodities. Another alternative is to degrade mycotoxins into non-toxic metabolites by using biotransforming agents such as bacteria or enzymes. Biotransformation has proven to be effective in detoxifying non-absorbable mycotoxins by altering their molecular structure and forming non-toxic metabolites which are then excreted. Studies have shown that plant-based products with antimicrobial properties may also have decontamination effects. The potential application of plant extracts, essential oils or isolated compounds in mycotoxin detoxification has been explored over the last few years. Cinnamon bark essential oil has been reported to effectively degrade fumonisin. Lemon and grapefruit oils have induced zearalenone degradation, and lemon and palmarosa oils have promoted a reduction in deoxynivalenol content. Aqueous extracts of rosemary, oregano and Corymbia citriodora, also known as lemon eucalyptus, have demonstrated the ability to degrade aflatoxin. In some of these works, the detoxifying effect has been proven by analyzing changes in the molecular structure of mycotoxins. Such studies evidence the great potential of chemical methods based on aqueous plant extracts and essential oils to degrade mycotoxins in contaminated food and feed products. However, they also report that effectiveness in mycotoxin degradation depends upon factors such as duration of contact between mycotoxin and detoxifying agent, temperature, pH and concentrations of both extract/essential oil and toxin. Although some investigations have demonstrated the efficacy of incorporating plant-based products as alternative food antioxidant and antimicrobial methods, their application as mycotoxin modifiers is limited to in vitro assays.

Benefits to fish health Although several studies address the benefits of using PFAs as health and growth promoters in fish, there is a paucity of information regarding the advantages of adding these additives to mycotoxincontaminated fish feed. The few (and recent) existing studies are focused on the use of PFAs to mitigate the noxious effects of aflatoxin B1-containing diets (Table 1). Limitations and perspectives The future of using plant-derived products in animal feed greatly depends on the information regarding practical plants features, value and chemical structure of the different molecules. The main limitations of using such products, especially in their unprotected form, lie in the instability and the strong aroma and flavor which may restrict applications. Encapsulation technologies can be assessed to provide controlled release of the phytogenic compounds, both in the animal feeding stuffs and gastrointestinal tract, taking into account the possible interactions with foods and/or fish intestinal environments. Additionally, there is a multiplicity of plants and different molecules to be explored as mycotoxin modifiers which may be more stable as well as add less odor and flavor to food. An integrated, multi-stage approach combining myriad actions should be used to tackle the mycotoxin problem. Plant-based natural products can be applied as antifungals, antioxidants and toxin modifiers in different levels of the aquaculture value chain. The combined effects of phytogenic feed additives and other binders or modifiers must also be explored. References available on request.

More information: Fernando J. Sutili Researcher, Fish Health and Nutrition Eloaqua, Brazil E: consult@eloaqua.com

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Improved DNA-analyses as tools to cover the need for evidence-based transparency in the aquaculture industry Erik Fuglseth, ORIVO

A sustainable industry The past decade has seen sustainability become a key driver in the aquaculture industry. It is well recognized that if the industry is to grow, it needs to do so in a sustainable manner. Practically all companies in the industry today claim to be acting sustainably, often referring to some of the established sustainability standards such as ASC, MSC or BAP. So far, this has been welcomed by seafood consumers. Recently, however, a shift in the behavior of these consumers has taken place. They now have a need to be reassured that there is truth behind the sustainability claims. Modern consumers are demanding an aquaculture industry that is more transparent about sustainability.

Transparency to support sustainability Sustainability claims are only as valid as the documentation backing it up. That is why traceability systems have been developed. Historically, such systems have been based on paper-based documentation, but current best practice entails fully digitalized systems based on secure technological solutions such as blockchain. In the seafood industry, these modern traceability systems are typically utilized by large vertically integrated seafood companies. Most of these systems make it possible to trace a fish filet all the way back to the egg the fish came from or to the GPS coordinates of the location where it was caught. Such systems are great tools to securely store

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and distribute data about a certain product, but there is one key requirement: the input needs to be valid. If that is not the case, false information will be spread about the product, established as the truth. Computer scientists call this GIGO, Garbage In, Garbage Out. So, if you can´t trust the data in a blockchain, you might as well go back to the old paper-based systems. This is where transparency comes to the rescue. By feeding data validated by independent third parties into a system, consumers can trust the information on a whole different level. This is true transparency, and the most valuable data of this sort comes from testing the consumer-ready products. This makes product adulteration close to impossible.

DNA-based analysis in the aquaculture industry All sustainability and traceability documentation should be backed up by evidence. For claims related to the origin of products such as fish filets or feed ingredients, laboratory analysis results represent excellent evidence. DNA-based analyses have been applied in the aquaculture industry for a number of years already. For aquaculture feed, in particular, PCR methods have been used to document the presence or absence of certain species in feed-related products. However, these methods have only been useful to a limited extent. Challenges related to falsepositive detections, low quantifiability in complex and/or processed mixtures, often combined with

high costs, have prevented the establishment of an industry-wide analysis standard. But the exponential growth in computer processing power experienced during the last decade has revolutionized the field of DNA analyses. A set of new analysis methods have emerged. They are referred to as Next Generation Sequencing (NGS), and they will have a big impact on the aquaculture feed industry in the years to come.

Next Generation DNA-Sequencing - NGS NGS, often referred to as high-throughput sequencing, is a term used to describe a set of several DNA-analysis methods. Common for all of them is that they are based on modern sequencing technologies making a direct determination of DNA sequences possible. In other words, they make it possible to “read” all the genetic information present in a given sample with high accuracy. This has also been possible in the past, but NGS has revolutionized the speed and cost at which it can be performed. A key advantage of NGS is that less DNA is required for each analysis. This is important for several reasons, one being that it makes it possible to avoid, or limit to a bare minimum, the step in the analysis protocol where DNA is amplified prior to the sequencing and species detection steps. This DNA-amplification step has been the core of many of the challenges of conventional DNA analyses, exemplified by the detection of false positives previously mentioned.

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customer and supplier is no longer good enough for consumers who are demanding evidence. As mentioned, laboratory analysis results represent such evidence and should, as a consequence of the recent development of NGS, become standard tools applied in the industry for important areas like ensuring sustainable sourcing of ingredients and supply-chain risk evaluations. The analysis results are excellent as input in digital traceability systems like blockchains, to form the basis of sustainability claims made on consumer products or company webpages.

By utilizing state-of-the-art big data analysis, NGS is also paving the way for a set of new application areas for DNA analyses in the aquaculture industry. With the proper experimental design and data-analysis setup, not only can false-positive results be avoided, but new features, like species quantification with unprecedented accuracy, be enabled. With NGS, DNA-based analyses are becoming a highly versatile tool. However, with the development of such powerful analysis methods comes a new set of challenges. The black-box issues of artificial intelligence-based analyses seen in other application areas, such as for self-driving cars, are valid also for NGS. The interpretation of raw data to understandable results, referred to as bioinformatics, requires highly specialized competence. Often, this competence also needs to be complemented with species and/or industry-specific knowledge to interpret the data correctly. Special care should therefore always be taken when developing and deploying NGS analyses for new application areas.

Adding value to the industry NGS analyses have already been commercially applied in parts of the aquaculture feed industry. Specifically, BioMar, the innovative Danish feed producer, is using a semi-quantitative version of NGS offered by ORIVO to screen the raw materials they are sourcing. The aim is to ensure that they know the origin of the ingredients they are buying and thereby avoid unwanted species in the feed. Because of the increased complexity in the marine ingredients value chain, the feed industry, in general, is experiencing increased scrutiny when it comes to documenting sustainable sourcing. Trust between

What does the future hold? There is no doubt NGS will be widely applied in the aquaculture industry in the future. Alone, or in combination with other novel innovative technologies, the potential application areas are numerous. Examples include determining catch areas for products coming from fisheries with strict geographic regulations, testing if a salmon filet is organic or not, and rapid on-site testing capabilities to determine the species composition of processed seafood products, utilized for instance by retail chains. Some of these applications require further technology development, and progress is already being made. With funding from the Norwegian Research Council, BioMar and ORIVO have recently, together with several highly recognized research institutions, embarked on a project to develop NGS-based methods able to accurately quantify the species composition in fishmeal and fish feed. Such work will surely bring exciting opportunities in the future, but NGS has already made significant contributions to our industry´s mission to provide consumers with evidence-based transparency. This consumer need is growing, and it has to be addressed throughout the value chain. References available on request.

More information: Erik Fuglseth CTO ORIVO AS E: efu@orivo.no

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Industry Events

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VOL 13 ISSUE 2 2021

Editorial: editor@aquafeed.com Editor: Lucía Barreiro Executive Editor/Publisher: Suzi Dominy Technical Editors: Peter Hutchinson, Albert Tacon, Ph.D Technical Manager: Marissa Yanaga

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